The long-term objective of this project is to understand the mechanisms of intracellular protein folding. Recent findings suggest that protein folding in vivo may generally involve assistance or catalysis by certain constitutively-expressed stress proteins termed molecular chaperones. This proposal focuses on the functions of Hsp70 and Hsp60 family members in this process. By studying protein folding reactions in mitochondria and with pure chaperone components, we have discovered the role of the mitochondrial Hsp60 and its bacterial homolog GroEL in mediating the folding of monomeric proteins. We have now reconstituted an ordered folding reaction in which the Hsp70 DnaK and GroEL act successively on folding polypeptides. The stress proteins DnaJ and GrpE function as coupling factors. The in vivo significance of this reaction is underscored by the finding that folding of proteins imported into mitochondria involves interaction with Hsp70 and subsequent transfer to Hsp60. Building upon this foundation, we propose to definitively establish the sequence, structural specificities and functions. of the interactions with chaperone proteins required for the correct folding of nascent and newly-synthesized polypeptides. We will test the hypothesis that there is a general protein folding pathway involving molecular chaperones of the Hsp70 and Hsp60 classes, or functional equivalents, in different cellular compartments. The following Specific Aims will be addressed in a multi-disciplinary experimental approach: 1.) Define the interactions between folding polypeptides and chaperone proteins in a reconstituted system containing pure DnaK, DnaJ, GrpE and GroEL/ES. The differential binding specificities of stress proteins for a series of intermediates of protein folding will be established. 2.) Define the sequential action of chaperones with folding polypeptides imported into intact mitochondria and translated in a cell-free system that can be depleted of endogenous chaperones. Luciferase will be used as a model protein whose activity can be assayed following in vitro synthesis. Protein folding intermediates and modified stress proteins will serve as tools. 3). Characterize the functions of stress proteins in the renaturation of thermally-denatured proteins in a reconstituted system and in intact mitochondria. 4). Discover novel molecular chaperones in eukaryotes, including DnaJ and GrpE homologs. The cytosolic protein complex Tcp1 will be isolated and assayed for an Hsp60-like activity in protein folding.